T4 dna ligase kit

A 1,442-bp 5′-flanking region and 48-bp exon 1 of kiss2 was obtained from a pair of primers containing two different restriction enzyme sites, respectively, namely, KpnI and XhoI. PCR was performed with PrimeSTAR HS DNA Polymerase (Takara, Tokyo, Japan). The PCR products and pGL4.10 vector (Promega, WI, USA) were digested by KpnI and XhoI restriction endonucleases (NEB, MA, USA). After purification, the digested products were ligated using T4 DNA Ligase Kit (NEB, MA, USA). The construction of recombinant vector above, namely, pkiss2-1442, was used as template to construct a series of deletion vectors, namely, pkiss2-912, pkiss2-775, pkiss2-660, pkiss2-433, and pkiss2-335. All constructs were sequenced ensuring accuracy. The recombinant vectors were extracted with Omega Endo-Free plasmid DNA mini kit II (OMEGA, GA, USA). Primers used in here are presented in Supplementary Table S1.

Genomic DNA was extracted from cell pellets using a QIAGEN QIAamp DNA mini kit and sheared using a Covaris Ultrasonicator (E220) to ~300 bp fragments. DNA concentration was measured using Qbit 4.0 reagents (ThermoFisher), and 200 ng of fragmented DNA was used for library preparation. End repair and A-tailing was carried out with NEBNext End repair reaction enzyme mix and buffer (E7442), and KAPA dual-indexed adapters (Roche) were ligated using the T4 DNA ligase kit from NEB (M0202). Post-ligation size selection was performed with AMPure XP beads (Beckman Coulter) before washing two times in 80% ethanol. Libraries were amplified using KAPA HiFi HotStart ready mix (Roche) and P5 and P7 primers (IDT). PCR program was as follows: 98 °C for 45 s, five cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. A further size selection and washing step was carried out after library amplification, and library quality was confirmed on Bioanalyzer chips (Agilent) and using a KAPA Library Quantification kit (Roche). Libraries were pooled and submitted for sequencing on NovaSeq 6000 at the New York Genome Center.

Streptavidin-HRP [N100, 1:4,000 for western blotting (WB)] and Dynabeads MyOne Streptavidin C1 (#65001) were purchased from Thermo Fisher Scientific. Rabbit anti-biotin antibody (ICP0611) was purchased from ImmuneChem. An anti-mCherry antibody was obtained from Novus Biologics (NBP2-25157). A mouse monoclonal anti-Flag antibody (no. F3165) was purchased from Sigma. Secondary antibody for immunofluorescence staining, anti-rabbit Alexa fluor 488, was obtained from molecular probes (1:1,000). Secondary antibodies for western blot, goat anti-rabbit IgG-HRP (sc-2004, 1:2,000) and goat anti-mouse IgG-HRP (sc-2005), were purchased from Santa Cruz Biotechnology. Enhanced chemiluminescence (ECL) western blotting substrate was obtained from Thermo Fisher Scientific (#32106). KOD Hot Start DNA polymerase (#71086-4) was purchased from Sigma. The T4 DNA ligase kit (#M0202M) and NEBuilder HiFi DNA Assembly Master Mix (M5520AA) were purchased from New England BioLabs.